Production of thyroxine from diiodotyrosine



Feb. 10, 1948. c. w. TURNER ETAL 2,435,947

PRODUCTION OF THYROXINE' FROM DIIODOTYROSINE' Filed Nov. 2, 1945THVAOX/NE MOLATED /0 40 Y 0 8O I00 TEMPERATURE -C" '47" GENE).

Patented Feb. 10, 1948 PRODUCTION OF THYROXINE FROM DIIODOTYROSINECharles W. Turner, Columbia, Mo., and Ezra P. Reineke, East Lansing,Mich., assignors to American Dairies Incorporated, Kansas City, Mo., acorporation of Maryland, and The Quaker Oats Company, Chicago, 11]., acorporat tion of New Jersey, jointly Application November 2, 1945,Serial No. 626,424

13 Claims.

This invention relates to improvements in the production of thyroxinefrom diiodotyrosine and refers more particularly to a method by which anincreased amount of high quality biologically active thyroxine isproduced from the originating substance. This application is acontinuationin-part of Serial No. 496,952, filed January 31, 1943, nowPatent No. 2,422,938, issued June 24, 1 947, and Serial No. 589,248,filed April 19, 1945. It has been known in the art that traces ofthyroxine can be produced from diiodotyrosine and research workers inthis field have incubated the product in mildly alkaline solutions attemperatures of the order of 37 C. Increased yields have been reportedby adding hydrogen peroxide at steam bath temperatures, then mixing withnormal butanol to extract the thyroxine as formed.

The instant procedure is an improvement over what has gone before andsubstitutes for conventional practice additional steps, the use of acatalytic material and the incorporation of oxygen during theincubationperiod to obtain an increased yield of the product. Laboratorytests indicated that when suflicient iodine wascombined with a proteinto substitute two atoms per molecule of tyrosine, the amount ofthyroxine formed can be influenced by the temperature of incubation andthe presence of a selected catalyst. This leads to the belief thatconditions favorable for maximum formation of thyroxine in iodinatedproteins would also influence the formation of thyroxine fromdiiodotyrosine.

In experiments conducted to determine the effect of these factors 21.6grams (0.05 mol.) of diiodotyrosine were dissolved in 700 ml. ofalkaline solution and incubated for 18 to 20 hours at the selectedtemperature. However, satisfactory results can be obtained withincubation periods varying from hoursto 72 hours or more to obtain thedesired results. The solutions were stirred continuously by means ofstirring motors adjusted to approximately 600 R. P. M. In thepreliminary experiments an attempt was made to determine the mostdesirable medium for the conversion of diiodotyrosine to thyroxine. Todo this,'results obtained with N/l-sodium hydroxide,

N/lO-sodium hydroxide and 7% sodium carbonate (NazCOs) were compared. Inall cases 21.6 grams of diiodotyrosine were dissolved in 700 ml. of eachsolution and incubated at70 C. with vigorous stirring for hours. Underthese conditions diiodotyrosine was highly stable in N/L-.sodium'hydroxide (NaOH), only traces of acidinsoluble material beingformed. Crystalline 2 thyroxine was recovered after incubation in boththesodium carbonate and N/10sodium hydroxide solutions. Considerablymore oxidative side reactions appeared to occur in the sodium carbonate(NazCOs) than in the N/lO-sodium hy-' droxide (NaOH) medium,'since theacid-insoluble precipitate obtained was darker and a smaller yield ofthyroxine was obtained. Thyroxine formation will occur with a broadrange of concentrations of both diiodotyrosine and sodium hydroxide inthe solutions incubated so long as the two are combined in suchproportions as to hold the pH of the solutions within the range ofapproximately 8.5 to 11. For instance, if 20 gm. of diiodotyrosine areused, approximately 700 ml. of one-tenth normal, or 70 ml. of one normalsodium hydroxide is required to maintain the requisite conditions forthyroxine formation. Subsequent preparations were incubated in N/10-sodium hydroxide solutions and the effect on thyroxine formation ofvarious factors was determined. Under the conditions used the solutionsremained at a pH of approximately 9.4 to 9.6 throughout the process. i

Investigations were then conducted to determine the influence and effectof incubation temperature on the amount of thyroxine formed under theconditions selected, and the effect exerted by a catalytic material suchas manganese oxide (M11304). To answer these questions diiodotyrosinesolutions were incubated at varying temperatures within the range of 40C. to 94 C. After incubation each solution was subjected to an isolationprocedure hereinafter described. At each temperature interval one samplewas incubated at N/lO-sodium hydroxide alone; to a second sample,otherwise treated identically with the first, 2 grams of manganese oxide(M13304) was added.

The drawing whichaccompanies this specification shows graphically thepercentage of thyroxine isolated at the temperatures employed, the fullline of the graph indicating recoveries of thyroxine when usingthecatalyst, the dotted line recoveries when the catalyst was not used.

At 40 C. only a trace of thyroxine was formed amounting to gross yieldsof 0.03% and 0.04%. With both types of treatment thyroxine formationincreased rapidly with increasing temperature until the optimum of C.was reached. Thereafter there was a decline in the amount of thyroxinerecovered, with zero recovery at 94 C. Throughout the effectivetemperature range there was an increased recovery of thyroxine 3 fromthe samples incubated in the presence of manganese oxide (MnaOr).

The maximum yield of thyroxine obtained at the optimum incubationtemperature of 60 C. was 183 mg, equivalent to a gross yield of 0.85%.However, of the diiodotyrosine initially processed, all but 6.5 grams onthe average can be recovered after separating the thyroxine. Based onthis figure the net yield of thyroxine is 2.8%.

In addition to the variable results with respect to yield illustrated bythe graph, the incubation system employed provided an opportunity tostudy the effect of such factorsasj 'the amount of stirring and aerationon the process of thyro'x'ine formation. Some of the preparations wereaerated continuously during incubation by bubbling finely dispersed airthrough them. Both the stirring and aeration were omitted in others,although the surface of the solutions was con stantly in contact withtheatmosphere. Other conditions being held constant, practicallyidenticalyieids of thyroxine 'were obt'ained where the solutioiis werestirred vigorously or aerated as indicated in table below. Withthestirring and aeration omitted, only traces of thyroxine were recovered;Thi's 'was true even when the usual amount or cat'alyst wa's added.Thus, it is evident that the formation'of thyroxine can be brought aboutunder these conditions by the incorporation at atmospheric oxygen.Manganese oXid'eslMriaO f will effectivelypatalyze the reaction but thecatalyst is ineffective in the absence of o 'ryge'n. In addition toMnsOr; other manganese compounds,- l'nclu'ding MnSOr, MnO, MnzOs, or themixture or manganese oxides formedby the reduction of KMnOr withglucose, effectively catalyze the -'racti'on.

. TABLE Efiect of stirring and aeration on formation of thyro'zrine fromdiiodotyrosz'ne Incubation Tempera- I ture, 0.

Procedure Mn's04,f2 g n; stirred at 600 R. P. M. M11304, 2 gm.; aeratedvigorously. ,Mu ol, 2 gun; stirred at 600 R.,P. M.

M1130 2 5111.; aerated vigorously.

Miis04, 2 gm.;-n'o.stirring or aeration. No catalyst; Stirred at 600 R.P. M. No catalyst; nostirring or aeration; M1304, 2 gm.; stirredvigorously.

We have also found that 'theconcentration of catalyst used is notcritical, satisfactory results being obtained when manganese catalyst inamounts equivalent to 0.1% to by Weight of the diiodotyrosine usedi'sadded.

The thyroxine formed during the incubation period may be protected bythe use-of varying quantities of n-butyl alcohol'as suggested in our ingat once. A part of the unaltered diiodotyrosine usually crystallizes outof the supernatant solutionafter standing for afew hours. In some of thelater tests in which it was desired to recover the unchangeddiiodotyrosine as completely as possible, thelf cidfinsoluble materialwas precipitated by dilute hydrochloric acid (H01) directly to theundiluted reaction mixture in an amount sufficient to redissolve thediiodotyrosine that crystallized at about pH 5.0. The acidinsolublesubstance still remained precipitated and could be recovered bycentrifuging. The diiodotyrosine was recovered from the supernatantsolution by first adding saturated sodium hydroxide (NaQH) to make itslightly alkaline and then aeidiifiying with glacial acetic acid(CI-IsCOOH), whereupon the :diiodotyrosine crystallized at once. Ineither 'ease-the acid-insoluble precipitate olved; 111300 ml. of N/10-sodium hydroxaOI-l') solution and then sufiicient dilute "c acid(HCl) was added to produce y color when the solution was tested withbromcresol-green indicator.

7 The thyroxirie was extracted by shaking the acidified solutionsuccessively with 300 ml. and

itate formed. 'Theprecipitate washed several time's with-5% a'c'e'ticacid ('ci-nooon) solu 'tion and-ana1'1'ye s e1vee ins minimum or boilmgsodium carbonate (Naz OOa) solution. The precipitate was mixed with 5ml; to 15 ml. of N/IO s'odium carbonate 'CNa'zCOQ' solution and thensaturated sodium carbonate (-NZCOB) was added dropwise in an amount justsufficient to dissolve the precipitate. A heavy whiteprecipitat'e of themohosodium saltof-thyroxine usually appear-ed immediately whenv thesolution was cooled. v r The solution was left; in therefrigerator overnight, andthen 'the-monosodium' thyroxine was recovered-bycentrifuging,dissolved in 70% alkaline-alcohol; and centrifuging: to remove the traceof undissolvedmaterial. When a few drops 01 glacial acetic acid(CH'aCOOH) were. added to the boiling solution, thyrox-ine crystallized.at once in the-typical' bundles of microscopic-needles.

A- totalof 2012 grams of thyroxine. obtained from these experiments waspooled and recrystallized twice from sodium carbonate (Macon-sclution.The pure-white monosodium salt. was

then dissolve in '70%- alkaline alcohol and free thyroxine fwa's-erysta-llized from the boiling solut-ion by'the-add'it'lon of'a'ceticacid (CH3COOH'). As suggested, thyrosrme appeared at once as the typicalbilndlesof microscopic "needles 'in ayield of 1.416 grams. The meltingpointwhen heated at the rate-0f 3 per minute was 230 C.-23=1 &C.Analysis showed 65.2%- iodine compared to the theoretical 65.4%.

Thus it winbeseen that a definite temperature optimi m for the remainsor; thyroirine from diiodotyrosine occurs'at"60? C. Incubation at 40C.,fth'e temperature commonly employed, yields onlyatrace' of 'thyroxinein a 20-hour period. likewise, the reactionis augmented by the catalyst.manganese oxide Minor); and other mange'nese con ipminds, whenoiry'gen-gis added. Thejimpqrtan'ce of atmospherie oxygen is evidentsince only trace of thyroxin'e were .ir rmed..ui s ai ,,v sx eweerfieeithe-selfi- 'tio'n' either bystirring-ondirectaeration. fliis is trueeven at the "optimum temperature and in the presence of the catalyst.

Workers in the art tion of thyroxine in iodinated protein by theoxidative coupling of two molecules of diiodotyrosine with theelimination of one side chain. The procedure recommended that oxidationbe brought about by the action of hypoiodite. If these teachings werefollowed, thyroxine formation should continue even in the absence ofadditional oxygen. From the results presented in table above, it isobvious that under the conditions used aeration of solutions isessential for the formation of appreciable amounts of thyroxine. Itthus. appears unlikely that hypoiodite plays an important role in thereaction.

From the fact that manganese will catalyze thyroxine formation only inthe presence of oxygen, it appears to act as an oxygen carrier for theoxidative coupling reaction that is apparently involved.

Thus it will be seen that, the invention is well adapted to attain theends and objects hereinbefore set forth, together with other advantageswhich are obvious and inherent to the process. There has been producedan increased yield of thyroxine from diiodotyrosine by control oftemperature to an optimum, the addition of oxygen and the use of aselected catalytic material.

It will be understood that certain features and subcombinations are ofutility and may be employed without reference to other features andsubcombinations. This is contemplated by and is within the scope of theclaims.

As many possible embodiments may be made of the invention Withoutdeparting from the scope thereof, it is to be understood that all matterherein set forth is to be interpreted as illustrative and not in alimiting sense.

Having thus described our invention, we claim:

1. A method of synthesizing thyroxine from diiodotyrosine comprising thesteps of incubating the diiodotyrosine in an alkaline solution withaeration at temperatures of 50 to 70 C. and extracting the thyroxinefrom the mixture.

2. A method of synthesizing thyroxine as in claim 1 in which aeration iseffected by stirring the solution with its surface exposed to anoxygen-containing gas.

3. A method of synthesizing thyroxine as in bating solution isN/lO-sodium hydroxide.

4. A method as in claim 1 in which the incubating solution is sodiumhydroxide having a pH of 8.5 to 11.

5. A method as in claim 1 in which the incuhave proposed theformabating'solution is a solution of sodium bicarbonate.

6. A method as in claim 1 in which aeration is effected by passing airthrough the solution.

7. A method of synthesizing thyroxine from diiodotyrosine comprising thesteps of incubating diodotyrosine in an alkaline solution with aerationat temperatures of 50 to C. in the presence of a catalyst and extractingthe thyroxine from the mixture.

8. A method of synthesizing thyroxine as in claim 7 in which anoxygen-containing gas is passed through the solution to effect aerationthereof.

9. A method as in claim 7 in which the incuhating solution isN/10-sodium hydroxide.

10. A method as in claim 7 in which the incubating solution is asolution of sodium bicarbonate.

11. A method as in claim 7 in which air is; passed through the solutionfor aeration.

12. A method as in claim 7 in which an oxide of manganese is used as thecatalyst.

13. A method as in claim 7 in which a mixture of the oxides of man aneseis used as the catalyst.

CHARLES W. TURNER. EZRA P. REINEKE.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 2,130,985 Lantenschlager et al.Sept. 20, 1938 2,309,404 Kraftet al Jan. 26, 1943 2,379,842 Turner etal. July 3, 1945 FOREIGN PATENTS Number Country Date 659,497 Germany May4, 1938 OTHER REFERENCES Chem, vol. 135, pp. 51-52

